1. Field
The present disclosure relates to methods of simultaneously transforming various types of nucleic acid sequences (e.g., genes) efficiently.
2. Description of the Related Art
Much biomaterials research is currently underway. Specifically, research into the production of industrially useful materials using microbes (i.e., microbial organisms) is in progress. However, microbial organisms do not only produce desired metabolites. When specific metabolites are produced in excess, growth of the microbial organisms may be inhibited, and the microbial organisms may no longer produce the desired metabolites or may produce undesirable by-products.
To overcome such limitations, research is underway to develop microbial organisms that may uniquely produce the desired metabolites. However, preparing transformed microbial organisms by transforming all metabolic pathways of the microbial organism requires a long period of time and great effort.
Specifically, the greater the number of nucleic acid sequences to be transformed, the greater the number of processes that are needed during the transformation. Each process is laborious and requires a long period of time, and gene transfer efficiency decreases. Accordingly, there is a need for improvement in gene transfer methods.
Microbial growth occurs regularly for a certain period of time, during which the microbes grow exponentially. However, after a certain period of exponential growth, the microbes show a slowed growth rate. When a population of the microbes is shown as a function of time, a typical S shaped curve may be obtained. A graph illustrating a number of the microbe population as a function of time is referred to as a growth curve of the microbes. The growth curve may be divided into four phases.
The first phase is a lag phase. The lag phase is a period in which the microbes adjust to a new medium. When the microbes are inoculated and cultured in the new medium, chemical changes such as an activation of enzymes and an increase in cell components such as RNAs occur. During the lag phase, the amount of RNA in cells increases substantially, the size of the microbes increases and respiratory activity of the microbes is high.
The second phase is a log phase. During the log phase, the microbes that passed through the lag phase grow actively. The log phase is a period in which the microbes grow logarithmically. As physiological functions of the microbes increase during the log phase, the microbes begin to excrete enzymes and become sensitive to physical and chemical treatments. Also, generation time and the size of the microbes are uniform during the log phase and the growth rate is determined according to environmental factors such as nutritive conditions, pH, temperature, and oxygen partial pressure of the medium.
The third phase is a stationary phase. The number of the microbes becomes constant after the log phase, the highest number of the microbes is maintained, and the microbes excrete enzymes, during this phase. Also during the stationary phase, some cells die while others grow, thereby maintaining a constant state where a number of dying microbes and a number of proliferating microbes are almost the same. Also, as the overcrowding of microbes causes depletion of nutrients, accumulation of metabolites of the microbes, changes in pH of the medium, and a depletion of the oxygen supply, the environment becomes unsuitable for growth of the microbes, and thus, the number of the microbes does not increase.
The last phase is a death phase. This is the final phase of culturing where the number of the microbes decreases. The primary reasons for death include dissolution of cells due to a decomposition caused by nuclease or protease, denaturing of enzymatic proteins, and autolysis of microbes killed by enzymes.
The microbes may be transformed by using microbes in an initial phase of the log phase, which is a period when the cells are most active and OD600 value is between about 0.5 to about 1.0. When the microbes subject to the transformation and the conditions of the transformation are controlled, an increase in the efficiency of the transformation may be observed.